The yield of many crop species is limited by the amount of nitrogen available in the soil, since nitrogen is a nutrient required for the synthesis of amino acids, proteins and other nitrogenous compounds such as DNA. To alleviate this limitation, farmers supplement the soil with nitrogenous fertilizers which are expensive and hazardous to the environment. Some important crop species such as the legumes soybean, pea, alfalfa, clover and bean, do not rely entirely on soil nitrogen but are able to meet their requirements by reducing atmospheric N.sub.2 to ammonia. This process is called N.sub.2 fixation and it is carried out by bacteria which form a symbiotic association with the roots of the host plant. The bacteria inhabit outgrowths of the root, termed nodules, which provide them with a suitable environment for N.sub.2 fixation. Biological N.sub.2 fixation is likely to become an essential component of sustainable agricultural systems, and a great deal of research is currently in progress to investigate the genetics of nodule formation and to determine the factors which regulate N.sub.2 fixation in leguminous crops. Consequently, a simple, accurate method is required for providing a non-invasive measurement of the rate of N.sub.2 fixation.
The reduction of atmospheric N.sub.2 to ammonia is catalyzed by the enzyme nitrogenase and the activity of this enzyme therefore determines the N.sub.2 fixation rate. Several methods have been devised to measure the N.sub.2 fixation rate and/or nitrogenase activity of legumes. These methods include:
(a) Measurement of whole plant nitrogen increment in which plants are harvested at different times during development and their nitrogen content measured. This method is destructive, labour intensive, requires complex and expensive equipment and does not distinguish between the nitrogen derived from the soil and that derived from N.sub.2 fixation. (b) Measurement of the .sup.15 N and .sup.14 N content of plant tissues and comparison of this with that of the atmosphere and the soil (the .sup.15 N natural abundance assay). Alternatively, nodulated roots may be fed with an atmosphere enriched in .sup.15 N.sub.2 (.sup.15 N.sub.2 enrichment assay) or a soil enriched in .sup.15 NO.sub.3.sup.- or .sup.15 NH.sub.4.sup.+ (.sup.15 N dilution assay) and the subsequent rate of .sup.15 N incorporation into tissues measured. These assays are destructive, time-consuming and require the use of expensive and complex analytical instruments. Also, like the nitrogen-increment method, they provide only a time-integrated measurement of nitrogen incorporation and do not show how nitrogenase activity and N.sub.2 fixation rate may vary over the short term.
(c) An acetylene reduction assay in which the nodulated roots of the legume are supplied with a gas containing about 10% acetylene and the reduction of this acetylene to ethylene is monitored with time. In the presence of 10% acetylene, virtually all electron flow through nitrogenase is diverted to acetylene reduction to ethylene. Therefore, the rate of ethylene production provides a measure of total nitrogenase activity. The assay can be performed by sealing the nodulated root into a closed cuvette containing 10% acetylene and then measuring ethylene accumulation measured with time (the closed system assay). Alternatively, 10% acetylene may be passed continuously through a cuvette containing the nodulated root, while ethylene concentration in the effluent gas stream is measured (the open system assay). In the former case, the assay provides only an isolated measurement of nitrogenase activity at a particularly time, while in the latter case taking discrete samples of effluent gas from the cuvette allows a time-course of nitrogenase activity to be measured. Both methods suffer from the fact that in vivo nitrogenase activity is inhibited by exposure of nodulated roots to acetylene. Consequently, the assays often greatly underestimate true activities. Also, the assays provide only a measurement of total nitrogenase activity and cannot be used to measure N.sub.2 fixation rate. In addition, the assays require the use of an expensive gas chromatograph, and the use of an explosive acetylene/air mixture that requires very careful handling.
(d) The H.sub.2 evolution assay which depends on the fact that during N.sub.2 fixation, the nitrogenase enzyme also reduces protons to H.sub.2 gas which is evolved from the nodule. H.sub.2 evolution rate may be measured by sealing a nodulated root in a cuvette and measuring the accumulation of H.sub.2 in the cuvette with time (the closed system H.sub.2 assay), or by passing gas through the cuvette continuously and monitoring H.sub.2 concentration in the effluent gas stream (the open system H.sub.2 assay). H.sub.2 may be monitored in discrete samples of the effluent gas by gas chromatography, or H.sub.2 concentration may be monitored continuously using a semi-conductor H.sub.2 analyzer such as that described by Layzell et al. (Plant Physiol. 582-585, 1984). The rate of H.sub.2 evolution in air provides a measurement of apparent nitrogenase activity (ANA) since only a proportion of the electron flow through nitrogenase is used for proton reduction. To measure total nitrogenase activity (TNA), it is necessary to expose nodulated roots to a gas mixture lacking N.sub.2, such as an Ar:O.sub.2 (80:20) mixture. In the absence of N.sub.2, all electron flow is diverted to proton reduction and the rate of H.sub.2 evolution from the nodule provides a measure of TNA (FIG. 1). The difference between the rates of H.sub.2 evolution in N.sub.2 :O.sub.2 and in Ar:O.sub.2 at a constant pO.sub.2 can be used to estimate N.sub.2 fixation rate thus: EQU N.sub.2 Fixation Rate=(TNA-ANA)/3 Equation 1
A denominator of 3 is used since 3 electron pairs are used in the reduction of N.sub.2 compared to 1 electron pair for the reduction of protons to H.sub.2 gas.
The measurement of H.sub.2 evolution using a H.sub.2 analyzer in the open system assay has several advantages over other methods for measuring nitrogenase activity and N.sub.2 fixation rate. These include:
The H.sub.2 analyzer is extremely sensitive and it is the only instrument which allows continuous, real-time measurement of nitrogenase activity. PA0 Measurements of ANA and short-term measurements of TNA are not inhibitory to nitrogenase so that measurements can be performed on the same plant material either continuously or intermittently over virtually any experimental period. PA0 Despite these advantages, relatively few researchers use the H.sub.2 evolution assay to measure nitrogenase activity. This is because the method has some disadvantages. These include:
The H.sub.2 analyzer is the only instrument which allows measurement of ANA, TNA, EAC and N.sub.2 fixation rate on the same plant material. PA1 The method is not labour intensive and the H.sub.2 analyzer is much cheaper than the mass spectrometer required for .sup.15 N measurements or the gas chromatograph required to measure ethylene production. PA1 The assay can only be used on legume symbioses which lack the enzyme uptake hydrogenase (HUP). This enzyme recycles some or all of the H.sub.2 produced by nitrogenase. Although H.sub.2 analysis cannot be used to measure nitrogenase activity in HUP.sup.+ symbioses, many agriculturally-important symbiosis are HUP.sup.-. PA1 Extended exposure of nodulated roots to Ar:O.sub.2 causes inhibition of nitrogenase. However, short-term exposures are not inhibitory and repeated assays of TNA can be made on the same plant material. PA1 The output of the H.sub.2 analyzer changes with pO.sub.2, with differences in water content of the gas stream and with the nature of the balance gas (N.sub.2 or Ar). The analyzer is, therefore, difficult and time-consuming to calibrate.
The acetylene reduction assay, and current methods of the H.sub.2 assay, require that plants are sealed in their growth pots to allow nitrogenase end-products to accumulate before analysis (closed system assays), or to allow nodulated root systems to be flushed with specific gas mixtures (open system assays). The procedures for both assays are time-consuming, and at least 5 minutes is required before stable measurements can be obtained. Also, the pots must be unsealed after the assays are completed if further growth and development of the plant is to be studied. This increases the time required to conduct the measurements, which limits the number of plants that can be assayed within a working day. A further limitation to the use of both the acetylene and H.sub.2 assays is that they require the use of sophisticated instrumentation, and the supply of specific gas mixtures to the material being studied. As a result the assays can only be conducted in laboratories within research institutions, or in mobile laboratories constructed in the field.
These limitations of the acetylene and H.sub.2 assays make them unsuitable for the large scale screening of plants that is needed to identify legumes with specific N.sub.2 -fixing characteristics. At present, much of the research in the field of N.sub.2 fixation involves genetic manipulation of legumes, and assessment of the effects of such manipulation on nitrogenase activity. This approach requires the growth of large populations of genetically altered plants (usually in a greenhouse) and assessment of their nitrogenase activities under various environmental conditions. To make appropriate statistical analyses of the data, the plants must be screened at the same stage of development and, as near as is possible, at the same time of day. It should be apparent, therefore, that there is need for a rapid method of nitrogenase activity analysis that can be performed in a greenhouse setting.